Identification of selective inhibitors of UGT1A3 and UGT1A8 and their application in UGT reaction phenotyping studies in human liver and intestinal microsomes

Uridine 5'-diphospho-glucuronosyltransferase (UGT) reaction phenotyping studies have posed significant challenges due to the limited availability of isoform-selective inhibitors. This recognized gap in reagent availability impedes the accurate determination of the contribution of specific UGT isoforms to the metabolism of UGT substrates. To address this challenge, 9 antibiotics were evaluated for their potential inhibitory effects on UGT isoforms. We identified 2 macrolide antibiotics, troleandomycin and erythromycin, as potent and selective inhibitors of UGT1A3 and UGT1A8, respectively. The mechanism of UGT inhibition by troleandomycin and erythromycin was investigated using recombinant UGT1A3 (mefenamic acid as probe substrate) and UGT1A8 (7-hydroxy-4-(trifluoromethyl)coumarin and apigenin as probe substrates). The results revealed a mixed-type inhibition mechanism, where troleandomycin and erythromycin allosterically inhibit UGT1A3 and UGT1A8, respectively. A slight positive cooperativity between erythromycin and substrate binding to UGT1A8 and a slight negative cooperativity between troleandomycin and substrate binding to UGT1A3 was observed. At saturating inhibitor concentrations, greater than 90% inhibition of glucuronidation catalyzed by UGT1A3 and UGT1A8 was observed. To validate these findings in human liver microsomes and human intestinal microsomes, telmisartan, a selective substrate of UGT1A3 and UGT1A8, was utilized. Similar to the results in expressed UGT isoforms, troleandomycin selectively inhibited UGT1A3 in human liver microsomes and erythromycin selectively inhibited UGT1A8 in human intestinal microsomes. The identification of these UGT isoform-selective inhibitors provides researchers with important new tools expanding the utility of in vitro UGT reaction phenotyping studies. SIGNIFICANCE STATEMENT: Identification of uridine 5'-diphospho-glucuronosyltransferase (UGT)1A3 (troleandomycin) and UGT1A8 (erythromycin) selective inhibitors addresses an important and heretofore unmet need for in vitro reaction phenotyping studies by facilitating the determination of the contribution of these enzymes to drug glucuronidation in humans.